Reconstitution for lab use
How to dissolve lyophilized peptides for in-vitro work: predicting solubility from charge and hydrophobicity, choosing between sterile water, bacteriostatic water, dilute acetic acid and DMSO, mixing gently, aliquoting and calculating stock concentrations.
Scope of this guide
This guide covers preparation of peptide stock solutions for laboratory experiments such as biochemical assays, cell culture and analytical work. It addresses solvent selection, dissolution technique, aliquoting and concentration calculations. It does not cover, and should not be read as guidance for, preparing anything for administration to people or animals.
The solvent should be chosen with the downstream experiment in mind from the start. A solvent that dissolves the peptide easily but is incompatible with the assay, such as a high DMSO concentration for sensitive cells or an unbuffered acid for an enzyme assay, simply moves the problem to a later step.
Before dissolving an entire vial, it is good practice to test solubility on a small portion of material, particularly for an unfamiliar sequence. A peptide that has been dissolved in the wrong solvent can sometimes be recovered by lyophilizing again, but this costs time and material.
Predicting solubility from the sequence
Solubility is governed mainly by net charge and hydrophobicity. To estimate net charge at neutral pH, count +1 for each lysine and arginine and for a free N-terminus, and −1 for each aspartic acid and glutamic acid and for a free C-terminal carboxyl. Histidine is only partially charged near neutral pH. Acetylation removes the N-terminal positive charge and C-terminal amidation removes the negative one.
Peptides with a clear net positive charge are basic, those with a clear net negative charge are acidic, and those near zero are neutral. Neutral peptides with a high proportion of hydrophobic residues such as Leu, Ile, Val, Phe, Trp and Met are the hardest to dissolve. The sequence analyzer reports net charge, isoelectric point and GRAVY hydrophobicity, which together give a useful starting estimate. Solubility is typically lowest near the isoelectric point.
Choosing a solvent
Start with the mildest solvent that is likely to work and that is compatible with the downstream experiment. A practical sequence is to try water first, then adjust pH or add a small amount of organic co-solvent if needed.
- Sterile water: the default first choice for short, charged or hydrophilic peptides. Use high-purity, sterile, endotoxin-free water when the solution will contact cells.
- Bacteriostatic water: sterile water containing benzyl alcohol as a preservative. It limits microbial growth in a solution that will be sampled repeatedly, but benzyl alcohol can be cytotoxic or interfere with some cell-based and biochemical assays, so plain sterile water or buffer is usually preferred for in-vitro work.
- Dilute acetic acid: for basic peptides that do not dissolve in water, a small amount of dilute acetic acid (commonly around 10 percent) protonates residual groups and improves solubility; dilute with water or buffer afterward.
- Dilute ammonium hydroxide or ammonium bicarbonate: the corresponding option for acidic peptides; avoid basic conditions for Cys-containing sequences, which oxidize faster at higher pH.
- DMSO: for hydrophobic or neutral peptides, dissolve first in a minimal volume of DMSO and then add aqueous buffer slowly while mixing. Keep the final DMSO concentration low enough for the assay, and consider DMF instead for Met- or Cys-containing peptides, since DMSO can oxidize them.
Dissolving the peptide
Allow the sealed vial to reach room temperature before opening, so moisture does not condense on the cold powder. Briefly centrifuge the vial so that all powder collects at the bottom. Add the solvent down the side of the vial rather than directly onto the powder.
Mix gently by swirling or slow inversion. Vigorous vortexing can cause foaming, which favors denaturation and aggregation at the air-water interface. If the material dissolves slowly, letting it stand for a few minutes or brief sonication in a water bath can help, while watching for warming. A clear solution with no visible particles or haze indicates dissolution; cloudiness suggests incomplete solubility or aggregation, and a different solvent may be needed.
For cell work, sterile-filter the final solution through a 0.22 µm low-protein-binding membrane where volume permits, keeping in mind that filtration removes microorganisms but not endotoxin; see endotoxin testing.
Concentration math
Stock concentration is the mass of peptide divided by the volume of solvent. For example, 5 mg of powder dissolved in 2.5 mL gives 2 mg/mL of powder. Because the powder also contains counter-ions and water, multiply by the net peptide content to obtain the true peptide concentration: with an NPC of 80 percent, the same solution contains 1.6 mg/mL of peptide. See purity vs net peptide content.
For molar concentration, divide the mass concentration in g/L by the molecular weight of the free peptide in g/mol. A peptide with a molecular weight of 1,000 g/mol at 1.6 mg/mL (1.6 g/L) is at 1.6 mM. To make a working dilution, use C1V1 = C2V2. These calculations, including NPC correction, can be done with the lab calculator.
- Mass concentration = peptide mass ÷ volume
- Corrected mass = powder mass × net peptide content
- Molarity (M) = concentration (g/L) ÷ molecular weight (g/mol)
- Dilution: C1V1 = C2V2
Aliquoting and storage
Once dissolved, divide the stock into single-use aliquots in low-binding polypropylene tubes, sized so that each experiment uses one aliquot. Label each tube with compound, lot number, concentration, solvent and date. Store aliquots frozen at -20 °C or -80 °C and avoid refreezing thawed portions.
Concentrated stocks are generally more stable and lose proportionally less material to surface adsorption than dilute solutions, so it is common to store a concentrated stock and dilute into assay buffer on the day of use. Record the solvent in the experimental notes, because DMSO, acetic acid or preservatives in the vehicle need to be matched in vehicle-control conditions. Further guidance is in peptide storage and stability.
Frequently asked questions
Should I use sterile water or bacteriostatic water for in-vitro experiments?
For most cell-based and biochemical experiments, sterile endotoxin-free water or a suitable buffer is preferred. Bacteriostatic water contains benzyl alcohol, which can affect cell viability and interfere with some assays. If a preservative is unavoidable, it should be included at the same concentration in vehicle controls.
My peptide will not dissolve in water. What should I try next?
Estimate the net charge from the sequence. For basic peptides, add a small amount of dilute acetic acid; for acidic peptides, a small amount of dilute ammonium hydroxide or bicarbonate. For hydrophobic or neutral peptides, dissolve in a minimal volume of DMSO, or DMF for Met- or Cys-containing peptides, then slowly add aqueous buffer.
How do I calculate the concentration of my stock solution?
Divide the mass of peptide by the volume of solvent, after multiplying the weighed powder mass by the net peptide content to correct for counter-ions and water. For molar concentration, divide the result in g/L by the peptide's molecular weight in g/mol. The lab calculator performs these steps.
Compounds mentioned
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